The complex functions of biological molecules are difficult to ascertain, in part because it is difficult to observe these molecules within functioning biological systems. Thus, the locus of a biomolecule's activity and the factors that actually interact with the biomolecule may not be apparent because it is difficult to distinguish one biomolecule from another within a living cell or tissue.
Labeled antibodies have been employed to identify specific factors within cells and tissues. But antibodies are large molecules that do not readily penetrate cells and binding of antibodies can often inhibit or modulate the functioning of the molecule to which it is bound.
Dyes have also been used to ‘color’ different cells and cellular factors. But researchers may not be able to distinguish one biomolecule from another, or trace the activity and functioning of a particular biomolecule, when using dyes because those dyes generally color many cellular structures and/or interrupt the functioning of the cells and/or biomolecules of interest.
Labeled antibodies and dyes also fail to provide sufficient signal strength to permit real-time observation of biomolecule activity. For example, while green fluorescent protein (GFP) has been used to observe the location of particular biomolecules within cells and/or tissues, GFP can require several hours to manifest fluorescence. Hence, the movements and interactions of GFP-linked biomolecules cannot be adequately traced in dynamic in vivo systems. Moreover, GFP cannot be used to observe several factors or biomolecules at once because GFP emits only one fluorescence color (green) and cannot be used to distinguish one biomolecule from another. GFP also requires oxygen, which is either not available or not plentiful in many cell types.
Therefore, new tools are needed that will permit real-time visualization of multiple biomolecules and factors at once.